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Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1
Author(s) -
Kawachi Miki,
Kobae Yoshihiro,
Kogawa Sayaka,
Mimura Tetsuro,
Krämer Ute,
Maeshima Masayoshi
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08613.x
Subject(s) - cytosol , chemistry , transmembrane domain , complementation , divalent , crystallography , antiporter , mutant , biophysics , biochemistry , amino acid , membrane , biology , gene , organic chemistry , enzyme
Arabidopsis thaliana MTP1 is a vacuolar membrane Zn 2+ /H + antiporter of the cation diffusion facilitator family. Here we present a structure–function analysis of At MTP1‐mediated transport and its remarkable Zn 2+ selectivity by functional complementation tests of more than 50 mutant variants in metal‐sensitive yeast strains. This was combined with homology modeling of At MTP1 based on the crystal structure of the Escherichia coli broad‐specificity divalent cation transporter YiiP. The Zn 2+ ‐binding sites of Ec YiiP in the cytoplasmic C‐terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in At MTP1. Although absent in Ec YiiP, Cys31 and Cys36 in the extended N‐terminal cytosolic domain of At MTP1 are necessary for complementation of a Zn‐sensitive yeast strain. On the cytosolic side of the active Zn 2+ ‐binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non‐selectively enhanced the metal tolerance conferred by At MTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn 2+ into the intra‐membrane Zn 2+ ‐binding site of At MTP1. A conformational change in the immediately preceding His‐rich cytosolic loop may displace Asn258 and permit Zn 2+ entry into the pore. This would allow dynamic coupling of Zn 2+ transport to the His‐rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn 2+ levels. Individual mutations at diverse sites within At MTP1 conferred Co and Cd tolerance in yeast, and included deletions in N‐terminal and His‐rich intra‐molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra‐ or inter‐molecular domain interactions, all of which are not conserved in the non‐selective Ec YiiP.